Methods for the quantitative analysis of organic compounds

Abstract

The invention comprises a low temperature method for the in situ derivatization and coupling of thermally and hydrolytically labile organic compounds such as spectinomycin to immunogenic carrier substances. The compound coupled to the immunogenic carrier is utilized to produce antibodies selective for the organic compound. Such methods allow for the quantitative analysis of the target organic compound and may be competitive or non-competitive immunoassays in which the dose response is directly or indirectly proportional to the concentration of spectinomycin-related substances. Such assays may be used to detect the presence of or to quantitate the amount of the organic compound in a complex and varied immunological background such as a sample of a biomass material. Detection of organic compound in the sample occurs through binding to a specific antibody. Amplification of that antibody-antigen response to increase the level of detection can be accomplished with a variety of markers producing either a fluorescent, ultraviolet, visible, chemiluminescent, gravimetric, amperometric, voltametric, or radioactive response.

Claims

1. A process for the chemical modification of an organic compound to form an immunogen comprising:

a) adding the organic compound containing either amine, hydroxyl or sulfhydryl functional groups to a homobifunctional carboxylic cross-linking agent in the presence of a hydroxylimide catalyst to form an adduct with a free carboxyl moiety;

b) activating the free carboxyl moiety of the adduct with a carbodiimide and eliminating a urea group to form an activated ester; and

c) conjugating the active ester to an immunogenic carrier containing a primary amine to form the immunogen.

2. The process of claim 1 wherein at least one of the steps of adding, activating or conjugating are carried out in a polar, aprotic organic solvent.

3. The process of claim 2 wherein the solvent is dimethylformamide, dioxane, dimethyl sulfoxide or acetonitrile.

4. The process of claim 1 wherein the organic compound is spectinomycin.

5. The process of claim 1 wherein the organic compound is dihydrospectinomycin.

6. The process of claim 1 wherein the homobifunctional carboxylic cross-linking agent is succinic anhydride.

7. The process of claim 1 wherein the catalyst is N-hydroxysuccinimide.

8. The process of claim 1 wherein the carbodiimide is dicyclohexylcarbodiimide.

9. The process of claim 1 wherein the active ester is the N-hydroxysuccinyl ester.

10. The process of claim 1 wherein the immunogen is a protein containing free amine functional groups or a protein in which free amine functional groups have been introduced through chemical modification.

11. The process of claim 10 wherein the protein is bovine serum albumin or ovalbumin or keyhole limpet hemocyanin.

12. The process of claim 1 wherein the immunogen is a polysaccharide modified to introduce free amine functional groups.

13. The process of claim 12 wherein the polysaccharide is dextran, sepharose, agarose or cellulose.

14. The process of claim 1 wherein the immunogen is a synthetic polymer or copolymer comprising polyacrolein or polyacrylamide or polyamide or polybutyrate or polyurea or polyureamide or polystyrene wherein a free amine is present or introduced as a result of modification.

15. The process of claim 1 wherein the adding, activating and conjugating steps are performed in less than about 4 hours.

16. The process of claim 1 wherein the adding, activating and conjugating steps are performed at about room temperature.

17. The process of claim 1 wherein the yield of immunogen from the process is greater than about 80%.

18. A process for the quantitation of an organic compound in a sample comprising:

a) forming an N-hydroxysuccinimide-derived (NHS) immunogen by a process comprising:

i) adding the organic compound to a homobifunctional carboxylic cross-linking agent in the presence of a hydroxylimide catalyst to form an adduct with a free carboxyl moiety;

ii) activating the free carboxyl moiety of the adduct with a carbodiimide and eliminating a urea group to form an activated ester; and

iii) conjugating the active ester to an immunogenic carrier containing a primary amine to form the NHS immunogen;

b) obtaining primary antibodies raised against the NHS immunogen of step a);

c) contacting the primary antibodies obtained in step b) with the sample to form primary antibody-organic compound complexes;

d) contacting the complexes of step c) to a solid support to which is attached NHS immunogen;

e) contacting secondary antibodies conjugated with detectable markers to the solid support; and

f) detecting secondary antibodies that bind to the solid support as an indication of the presence of the organic compound.

19. The process of claim 18 wherein the organic compound is spectinomycin or dihydrospectinomycin.

20. The process of claim 18 wherein the sample is a sample of biomass.

21. The process of claim 20 wherein the sample of biomass comprises soil, water, plant materials, animal fluids or animal tissues.

22. The process of claim 18 wherein the NHS immunogen comprises the organic compound conjugated to a carrier moiety.

23. The process of claim 18 wherein the detectable markers are enzymes, colored dyes, fluorescent dyes, chemiluminescent dyes, latex beads, magnetic particles or radioactive labels.

24. The process of claim 18 wherein the solid support is a microtiter plate or nitrocellulose membrane filter.

25. The process of claim 18 wherein detecting involves the addition of enzyme substrate, direct visual inspection, spectrophotometric measurement, fluorescent measurement, amperometric measurement, voltametric measurement, gravimetric measurement, exposure to a magnetic field or radioactive measurement.

26. A process for quantifying an amount of an organic compound in a sample comprising:

a) forming an N-hydroxysuccinimide-derived (NHS) immunogen by a process comprising:

i) adding the organic compound to a homobifunctional carboxylic cross-linking agent in the presence of a hydroxylimide catalyst to form an adduct with a free carboxyl moiety;

ii) activating the free carboxyl moiety of the adduct with a carbodiimide and eliminating a urea group to form an activated ester; and

iii) conjugating the active ester to an immunogenic carrier containing a primary amine to form the NHS immunogen;

b) obtaining antibodies raised against the NHS immunogen of step a);

c) contacting the sample with the antibodies obtained in step b) to form antibody-organic compound complexes;

d) contacting the complexes of step c) to a coating antigen fixed on a solid support; and

e) detecting bound complexes as an indication of the presence of the organic compound.

27. The process of claim 26 wherein the organic compound is spectinomycin.

28. The process of claim 26 wherein the organic compound is dihydrospectinomycin.

29. The process of claim 26 wherein the NHS immunogen is a conjugate of spectinomycin and keyhole limpet hemocyanin.

30. The process of claim 26 wherein the solid support is a microtiter plate or nitrocellulose membrane.

31. The process of claim 26 wherein the detectable markers are enzymes, colored dyes, fluorescent dyes, chemiluminescent dyes, latex beads, magnetic particles or radioactive labels.

32. The process of claim 26 wherein the detection step involves the addition of enzyme substrate, direct visual inspection, spectrophotometric measurement, fluorescent measurement, amperometric measurement, voltametric measurement, gravimetric measurement, exposure to a magnetic field or radioactive measurement.

33. A process for the detection of an organic compound comprising:

a) forming an N-hydroxysuccinimide-derived (NHS) immunogen by a process comprising:

i) adding the organic compound to a homobifunctional carboxylic cross-linking agent in the presence of a hydroxylimide catalyst to form an adduct with a free carboxyl moiety;

ii) activating the free carboxyl moiety of the adduct with a carbodiimide and eliminating a urea group to form an activated ester; and

iii) conjugating the active ester to an immunogenic carrier containing a primary amine to form the NHS immunogen;

b) obtaining antibodies raised against the NHS immunogen of step a);

c) immobilizing the antibodies of step b) to a solid support;

d) contacting the sample with the immobilized antibodies of step c) to form immobilized antibody-organic compound complexes;

e) contacting the immobilized complexes of step d) with detectable markers conjugated with the organic compound; and

f) detecting bound marker as an indication of the presence of the organic compound.

34. The process of claim 33 wherein the detectable markers are selected from the group consisting of enzymes, colored dyes, fluorescent dyes, chemiluminescent dyes, latex beads, magnetic particles and radioactive labels.

35. The process of claim 33 wherein the solid support is a microtiter plate or nitrocellulose membrane filter.

36. The process of claim 33 wherein the detecting step involves adding enzyme substrate, direct visual inspection, spectrophotometric measurement, fluorescent measurement, amperometric measurement, voltametric measurement, gravimetric measurement, exposure to a magnetic field or radioactive measurement.

37. The process of claim 33 wherein the organic compound is spectinomycin.

38. The process of claim 33 wherein the organic compound is dihydrospectinomycin.